Bottom Line:
Here we found that recognition of intracellular antibodies by TRIM21 activated immune signaling.Intracellular antibody signaling was abrogated by genetic deletion of TRIM21 and was restored by ectopic expression of TRIM21.Thus, the antibody-TRIM21 detection system provides potent, comprehensive activation of the innate immune system independently of known pattern-recognition receptors.

ABSTRACTDuring pathogen infection, antibodies can be carried into the infected cell, where they are detected by the ubiquitously expressed cytosolic antibody receptor TRIM21. Here we found that recognition of intracellular antibodies by TRIM21 activated immune signaling. TRIM21 catalyzed the formation of Lys63 (K63)-linked ubiquitin chains and stimulated the transcription factor pathways of NF-κB, AP-1, IRF3, IRF5 and IRF7. Activation resulted in the production of proinflammatory cytokines, modulation of natural killer stress ligands and induction of an antiviral state. Intracellular antibody signaling was abrogated by genetic deletion of TRIM21 and was restored by ectopic expression of TRIM21. The sensing of antibodies by TRIM21 was stimulated after infection by DNA or RNA nonenveloped viruses or intracellular bacteria. Thus, the antibody-TRIM21 detection system provides potent, comprehensive activation of the innate immune system independently of known pattern-recognition receptors.

Mentions:
TRIM5α has been shown activate AP-1 and NF-κB signaling pathways, both constitutively within the LPS stimulation pathway and upon infection with retroviruses, by catalyzing the formation of free K63-linked ubiquitin chains12. In the presence of K63-specific E2 enzymes UBC13 and UEV1A, we found that TRIM21 catalyzed K63-linked chain formation in vitro(Fig. 2a). Both TRIM21 and the ubiquitin E2 conjugation enzymes remained unmodified suggesting that the resulting chains are unanchored (Fig. 2a and Supplementary Fig. 4). Ubiquitin chain formation was observed with wild-type or K63-only ubiquitin but not K63R, confirming that linkage is through lysine 63 (Fig. 2b). Ubiquitination was dependent on the RING domain (Fig. 2c) and accordingly, TRIM21 signaling was RING dependent, as Trim21-deficient MEFs expressing human TRIM21 lacking the RING domain (ΔRING) failed to activate NF-κB upon challenge with AdV + Ab (Fig. 2d). However, TRIM21 signaling, but not in vitro ubiquination activity was dependent on the PRYSPRY domain (Fig. 2c, d). This is suggestive of a greater complexity of regulation of TRIM21 activation in cells. Depletion of UBC13 using siRNA substantially reduced signaling in response to AdV + Ab in both MEF and HeLa cells (Fig. 2e, f). This reduction was similar to that observed upon challenge with TNF (Supplementary Fig. 5), a finding which is in accordance with UBC13’s reported involvement in TNF receptor signaling14. Together, these data show that TRIM21 catalyzes K63-linked ubiquitin chain formation in a RING-dependent manner and that deletion of the RING or depletion of the K63 E2 conjugating enzyme, UBC13, prevents efficient signaling in response to infection with antibody-coated adenovirus.

Mentions:
TRIM5α has been shown activate AP-1 and NF-κB signaling pathways, both constitutively within the LPS stimulation pathway and upon infection with retroviruses, by catalyzing the formation of free K63-linked ubiquitin chains12. In the presence of K63-specific E2 enzymes UBC13 and UEV1A, we found that TRIM21 catalyzed K63-linked chain formation in vitro(Fig. 2a). Both TRIM21 and the ubiquitin E2 conjugation enzymes remained unmodified suggesting that the resulting chains are unanchored (Fig. 2a and Supplementary Fig. 4). Ubiquitin chain formation was observed with wild-type or K63-only ubiquitin but not K63R, confirming that linkage is through lysine 63 (Fig. 2b). Ubiquitination was dependent on the RING domain (Fig. 2c) and accordingly, TRIM21 signaling was RING dependent, as Trim21-deficient MEFs expressing human TRIM21 lacking the RING domain (ΔRING) failed to activate NF-κB upon challenge with AdV + Ab (Fig. 2d). However, TRIM21 signaling, but not in vitro ubiquination activity was dependent on the PRYSPRY domain (Fig. 2c, d). This is suggestive of a greater complexity of regulation of TRIM21 activation in cells. Depletion of UBC13 using siRNA substantially reduced signaling in response to AdV + Ab in both MEF and HeLa cells (Fig. 2e, f). This reduction was similar to that observed upon challenge with TNF (Supplementary Fig. 5), a finding which is in accordance with UBC13’s reported involvement in TNF receptor signaling14. Together, these data show that TRIM21 catalyzes K63-linked ubiquitin chain formation in a RING-dependent manner and that deletion of the RING or depletion of the K63 E2 conjugating enzyme, UBC13, prevents efficient signaling in response to infection with antibody-coated adenovirus.

Bottom Line:
Here we found that recognition of intracellular antibodies by TRIM21 activated immune signaling.Intracellular antibody signaling was abrogated by genetic deletion of TRIM21 and was restored by ectopic expression of TRIM21.Thus, the antibody-TRIM21 detection system provides potent, comprehensive activation of the innate immune system independently of known pattern-recognition receptors.

ABSTRACTDuring pathogen infection, antibodies can be carried into the infected cell, where they are detected by the ubiquitously expressed cytosolic antibody receptor TRIM21. Here we found that recognition of intracellular antibodies by TRIM21 activated immune signaling. TRIM21 catalyzed the formation of Lys63 (K63)-linked ubiquitin chains and stimulated the transcription factor pathways of NF-κB, AP-1, IRF3, IRF5 and IRF7. Activation resulted in the production of proinflammatory cytokines, modulation of natural killer stress ligands and induction of an antiviral state. Intracellular antibody signaling was abrogated by genetic deletion of TRIM21 and was restored by ectopic expression of TRIM21. The sensing of antibodies by TRIM21 was stimulated after infection by DNA or RNA nonenveloped viruses or intracellular bacteria. Thus, the antibody-TRIM21 detection system provides potent, comprehensive activation of the innate immune system independently of known pattern-recognition receptors.